Environmental cues associated with prior cocaine use are powerful triggers for relapse, even in users who have been abstinent for a long period of time. Efforts to understand this persistent vulnerability have revealed that, in rats and humans, cue-induced craving progressively intensifies (`incubates') during abstinence from cocaine self-administration. Our studies of incubation have focused on excitatory synapses in the nucleus accumbens (NAc) core, where GluA2-containing Ca2+-impermeable AMPARs (CI-AMPARs) normally mediate the bulk of AMPAR transmission. We found that strengthening of these synapses, via incorporation of higher conductance Ca2+-permeable AMPARs (CP-AMPARs), occurs after about a month of withdrawal from extended-access cocaine self-administration and thereafter underlies expression of incubated cocaine craving. By understanding the cascade leading to CP-AMPAR accumulation, we may identify novel targets for therapeutic intervention. The goal of this R21 is to test the hypothesis that two direct effectors of cyclic AMP - PKA (protein kinase A) and Epac (exchange protein directly activated by cAMP) - work together to increase the relative contribution of CP-AMPARs to synaptic transmission in the NAc during incubation. Specifically, we hypothesize that, during cocaine withdrawal, Epac activation in NAc medium spiny neurons (MSNs) leads to p38 MAPK activation and removal of CI-AMPARs; this ?makes room? for CP-AMPARs to enter these synapses via a mechanism involving PKA activation. In Aim 1, we will use whole-cell patch-clamp recordings to determine if postsynaptic Epac and p38 MAPK activation leads to removal of CI-AMPARs from NAc synapses in drug-nave male and female rats, and whether this effect is occluded after incubation of craving. If warranted, we will determine if shRNA knockdown of Epac2 in the NAc core prevents incubation of craving and CP-AMPAR accumulation. Aim 2 will test the effects of postsynaptic PKA activation on AMPAR transmission in MSNs of drug-nave rats and after incubation of craving, and assess the level of PKA activity in the NAc during incubation. This project is well suited to the R21 mechanism because Epac is a new focus for our lab and the field (a pubmed search on cocaine and Epac revealed only 3 papers) and because very little is known about the role of postsynaptic PKA signaling in regulating AMPAR transmission in NAc MSNs. Significance is high not only because of the need for novel therapeutic approaches to cocaine relapse but because our findings will provide insight into basic mechanisms regulating excitatory synaptic transmission in the NAc, which is relevant to many brain disorders. Finally, this work will set the stage for an R01 application testing the novel hypothesis that Ca2+ signaling in NAc MSNs decreases during cocaine withdrawal; ultimately this disinhibits adenylyl cyclase 5, the predominant isoform in MSNs, leading to Epac/PKA activation and CP-AMPAR insertion.